Toxicities associated with most preparative regimens for hematopoietic cell transplantation (HCT) and the risk of graft-versus-host-disease (GVHD) limit the applicability of mixed chimerism approaches for allograft tolerance. The overall objective of this proposal is to further develop a novel protocol for transient chimerism and tolerance induction that does not rely on toxic conditioning and avoids the risk of GVHD. Some miniature swine HCT recipients conditioned with this novel protocol develop transient peripheral blood chimerism that becomes undetectable within 1-3 months post transplantation, similar to the transient chimerism observed in patients. Preliminary data demonstrate that exposure to donor peripheral blood cells using this protocol results in stable B-cell tolerance together with transient T-cell tolerance, and that immune modulation mediated by allogeneic cells occurs independently from stem cell engraftment. As newly developing B cells are continuously forming throughout life in the bone marrow, the finding that donor cells do not persist and no other source of donor antigen is present makes it unlikely that clonal deletion mechanisms are solely responsible for maintenance of B-cell tolerance in this model. We hypothesize that an active regulatory mechanism is involved in preventing germinal center formation upon further exposure to donor cells and tissue and that use of this novel mild conditioning protocol preserves this regulatory mechanism. We also hypothesize that the immune modulatory response to allogeneic cells plays a direct role in facilitating organ allograft tolerance induction. We propos that, in contrast to current dogma, preserving rather than completely ablating host immune cells, including B- and T-cell populations, at the time of exposure to donor cells may be beneficial for inducing stable tolerance mechanisms. The goals of this research proposal are to determine the mechanisms and the necessary immunosuppressive conditions which lead to reliable induction of transplantation tolerance through transient chimerism in swine, and to translate these findings to non-human primates (NHP). These goals will be achieved through the following Specific Aims: 1) Determine the mechanisms involved in induction of stable B-cell tolerance and transient T-cell unresponsiveness following allogeneic cell infusion using MHC-defined miniature swine; 2) Determine the donor cell source and the necessary components of the recipient conditioning protocol which lead to consistent induction of immunomodulatory rather than immunostimulatory responses to allogeneic cells in swine; and 3) Confirm the efficacy of this novel approach for achieving stable renal allograft tolerance in both swine and NHP as a prerequisite to clinical trials. Results of these studies will guide further clinical protocol modifications to enhance allograft tolerance while significantly reducing toxicity in transplant patients.